This invention is related functional mediators of genes and functional organization of such mediators into pathways.
Many biological functions are accomplished by altering the expression of various genes through transcriptional (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle regulation, cell differentiation and cell death, are often characterized by the variations in the expression levels of groups of genes.
Gene expression is also associated with pathogenesis. For example, the lack of sufficient expression of functional tumor suppressor genes and/or the over expression of oncogene/protooncogenes could lead to tumorgenesis (Marshall, Cell, 64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991), incorporated herein by reference for all purposes). Thus, changes in the expression levels of particular genes (e.g. oncogenes or tumor suppressors) serve as signposts for the presence and progression of various diseases.
The study of gene expression in the art has been generally concentrated on the regulatory regions of the gene of interest and on the relationships among a few genes. A number of transcriptional factors/DNA binding proteins have been identified and a limited number of regulatory pathways have been discovered. However, the expression of a particular gene is frequently regulated by the expression of a large number of other genes. The expression of those regulatory genes may also be under the control of additional genes. This complex regulatory relationship among genes constitutes a genetic network. The function and regulation of a particular gene can be best understood in the context of this genetic network. As the Human Genome Project and commercial genome research progress at a great rate, most, if not all, of the expressed genes will be partially sequenced in the near future. Understanding the functions and regulatory relationships among the large number of genes is becoming a difficult task with traditional tools.
Communication through signal transduction pathways is usually achieved through a combination of transcriptional and post-transcriptional mechanisms. Because signaling events are causally linked within given pathways, genetic alterations that disrupt either of these two classes of regulatory mechanisms should ultimately affect gene expression.
There is a need in the art to develop a systematic approach to understand the complex regulatory relationships among large numbers of genes, in particular those involved in signal transduction.
It is an object of the present invention to provide methods of determining candidate functional mediators of genes.
It is another object of the present invention to provide methods for determining a pathway of gene products.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided for determining candidate functional mediators of a transgene. The method comprises: altering expression of a first selected gene in a first of two populations of identical cells. Expression levels of a set of genes in the two populations of cells are compared. Genes in the set whose expression levels differ between the two populations of cells are identified. The genes identified are candidate functional mediators of the selected gene. Expression of a second selected gene is then altered in one of a third and fourth populations of cells. The third and fourth populations comprise identical cells. The second selected gene is a candidate functional mediator of the first selected gene. Expression levels of a set of genes in the third and fourth populations of cells are compared. Genes in the set whose expression levels differ between the third and fourth populations of cells are identified. The genes identified are candidate functional mediators of the second selected gene.
According to another embodiment a method is provided for identifying pathways of functional mediators of a selected gene. The method comprises altering expression of a first selected gene in a first of two populations of identical cells. Expression levels of a set of genes in the two populations of cells are compared. Genes in the set whose expression levels differ between the two populations of cells are identified. The genes identified form a set of candidate functional mediators of the first selected gene. Expression of a second selected gene is altered in one of a third and fourth populations of cells which populations comprise identical cells. Expression levels of the set of genes in the third and fourth populations of cells are compared. Genes in the set whose expression levels differ between the third and fourth populations of cells are identified. The genes identified form a set of candidate functional mediators of the second selected gene. The candidate functional mediators identified of the first and second selected genes are compared. Genes which are identified as candidate functional mediators of both selected genes suggest that the first and second selected genes are components of a common pathway.
Failure to identify a candidate functional mediator of both selected genes suggests that the two selected genes are in different pathways. Identification of the set of candidate functional mediators of the first selected gene as a subset of the set of candidate functional mediators of the second selected gene suggests that the first selected gene is downstream in a pathway relative to the second selected gene. A candidate functional mediator which is identified in only one of the two sets of candidate functional mediators is identified as upstream in the pathway of a selected gene from a point of convergence with the pathway of the other selected gene, if the sets of candidate functional mediator genes of the first and second selected genes contain common members.
In still another aspect of the invention, a method is provided to determine a pathway of gene products. The method comprises comparing a first set of genes with a second set of genes. The first set is identified by comparing a first set of candidate functional mediator genes with a second set of candidate functional mediator genes. The first set is identified by the process of:
(a) comparing expression levels of a set of genes in two populations of identical cells, wherein a first of the two populations of cells has been treated to alter expression of a first selected gene;
(b) identify genes in the set whose expression levels differ between the two populations of cells, wherein the genes identified are candidate functional mediators of the first selected gene.
The second set is identified by the process of:
(c) comparing expression levels of the set of genes in a third and fourth population of cells, wherein the third population of cells has been treated to alter expression of a second selected gene;
(d) identifying genes whose expression levels differ between the third and fourth populations of identical cells, wherein the genes identified are candidate functional mediators of the second selected gene. The first and second selected genes are identified as components of a common pathway when one or more genes are found to be candidate functional mediators of both of said first and said second selected genes. Alternatively, the first and second selected genes are identified as being in different pathways when no gene is identified as being a candidate functional mediator of both of said first and said second selected genes. In another embodiment a gene which is found to be a candidate functional mediator of only one of said first and said second selected genes is identified as upstream in the pathway of the first or second selected gene from a point of convergence with the pathway of the second or first selected gene, if the first and second sets of candidate functional mediator genes contain common members. In still another embodiment the first selected gene is identified as downstream in a pathway relative to the second selected gene if the set of candidate functional mediators of the first selected gene is a subset of the set of candidate functional mediators of the second selected gene.
These approaches can be used to interrogate the genetic regulatory network and to construct a map indicating regulatory relationships.